Development of a Novel Anti-Adhesive Vaccine Against Pseudomonas aeruginosa Targeting the C-terminal Disulfide Loop of the Pilin Protein

Type IV pili (T4P) are major virulence factors of Pseudomonas aeruginosa (P. aeruginosa) that are associated with primary adhesion, biofilm formation and twitching motility. This study focuses on the introduction of a novel biologically active subunit vaccine derived from the disulfide loop (DSL) of P. aeruginosa pilin. We investigated the expression of the novel PilA in-frame with pET26b vector, which contains three domains, that each domain contains three tandem repeats. The flexible (GGGGS) and (GGGGS)3 linkers were linked between the three tandem repeats and each pilA domain, respectively. The recombinant construct (pET26b/pilA) was transformed and expressed in Escherichia coli BL21 (DE3). The reactivity of specific antiserum against PilA was assessed by ELISA method. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis and twitching inhibition assays. The pET26b/pilA plasmid was confirmed by enzymatic digestion. The purified PilA protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen was 1:8. The results of opsonophagocytosis assay revealed that the antibodies raised against PilA promoted phagocytosis of the PAO1 and 6266E strains to some extent (17.5% and 16.3%, respectively), so the twitching inhibition test confirmed this result. Taken together, these are the preliminary results based on a first chimerical structure failure to induce antibodies that promote the opsonization and eradication of the pathogen. Therefore, the biological activity of the PilA protein showed that it should be introduced with other proteins or target antigens against P. aeruginosa in the future studies.

he bacterium Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous organism that is also known as an opportunistic human pathogen, causing both chronic and acute infections in susceptible populations, including individuals with burn wounds or cystic fibrosis, or intensive care unit patients (1). P. aeruginosa can infect almost any part of the human body, but typically targets surface-exposed epithelial cells, such as in the skin, airways and eyes. The most important question that still remains unanswered for non-obligate pathogens like P. aeruginosa is how these bacteria initiate infections after entering the host. Given that P. aeruginosa is among the many bacteria that whereas non-piliated strains were reported to show a 90% decrease in their ability to bind human pneumocytes (5), and also mutant strains that are unable to produce T4P are attenuated in virulence (6,7). Furthermore, another study revealed that non-piliated strains caused 28%-96% fewer cases of P. aeruginosa pneumonia as compared to piliated strains in a mouse model of infection (8).
T4P plays an important role in many processes, including attachment to biotic and abiotic surfaces, DNA uptake, biofilm formation, phage transduction and a special form of bacterial cell movement, known as 'twitching motility' (9), hence, they have been used as an ideal target antigen for vaccine development.
T4P have been classified into two different subtypes, type IVa pilus (T4aP) and type IVb pilus (T4bP), based on differences in the architecture of the assembly systems and in the structure of the major pilin subunit. T4aP is found in a wide variety of bacterial species such as P. aeruginosa, whereas T4bP is found predominantly in enteric pathogens (3). The pilus fiber is composed of thousands of copies of PilA (or pilin, the major structural subunit) that are encoded by an operon that is positively controlled by the algR regulator (10).
The PilA monomer can be divided into three regions: a highly conserved hydrophobic Nterminal α-helix region; a hypervariable central region, and a semi-conserved C-terminal region containing β-strands. Pili binds buccal, tracheal and corneal epithelial cells, specifically through interaction with a disulfide-loop (DSL) region that is found at the C-terminus of the pilin monomer called the receptor-binding domain (RBD). This domain of P. aeruginosa pilin is a suitable candidate for a peptide vaccine (4). The DSL region is structurally highly conserved among type IV pilins of all species of P. aeruginosa, although the size of the loop (from 12 to 31 amino acids in P. aeruginosa) and its sequence are varied among pilin alleles. The C-terminal DSL of the pilin subunit was shown by monoclonal antibodies that mediate attachment to epithelial cell receptors, this finding suggests that PilA itself acts as both a major structural subunit and an adhesin (7,11). There are several studies mentioning that the attachment of P.
aeruginosa to the respiratory mucosa, skin, corneal T surfaces is multifactorial. P. aeruginosa type IV pili are thought to interact with the glycosphingolipid epithelial receptor asialo-GM1 (aGM1) via the DSL, which is proposed to be exposed only at the tip of pilus. The membrane distribution of asialo-GM1 is compared to that of caveolin-1 and understanding lipid partitioning between membrane domains (11,12). Finally, T4P from all strains of P. aeruginosa shares a common receptor. However, the sequence diversity presents a significant obstacle to the development of a broadly protective RBD-based vaccine targeting the T4P.
The pET-based vectors are the most powerful systems, yet developed for the cloning and expression of recombinant proteins in the E. coli.
One vector of this system, pET26b(+) (abbreviated as pET26b), was used in this study for the construction and expression of recombinant proteins. This expression vector is under the control of the T7 promoter and carries an N-terminal PelB signal sequence for potential periplasmic localization of recombinant protein and optional Cterminal His-tag sequence for detection and purification (13). In the present study, we designed a chimeric plasmid that contains pilA region, which encodes the three peptides of DSL (in triplicate forms). The pilA region contains three domains,

Construction of the expression vector
The pilA gene was inserted into the E. coli

Expression and isolation of the recombinant protein
The expression and purification of the r-PilA protein was performed as previously described (14), was transformed with the plasmid pET26b/pilA. A single colony was grown overnight in 5 ml LB medium supplemented with kanamycin (30 µg/ml)  finally aliquoted in 0.5 mg/ml vials.

Immunoblot analysis
The purified r-PilA was electrophoresed in a

Enzyme-linked immunosorbent assay (ELISA)
To detect the humoral immune responses

Opsonophagocytic killing assay (OPA)
The ability of antisera to opsonize P.
aeruginosa for killing in the presence of complement and macrophage was evaluated using an in vitro OPA as described previously (15).

Twitching inhibition test
To verify the functionality of the specific polyclonal antibody, the twitching inhibition assay was carried out according to Castric

Statistical analysis
Data were analyzed by ANOVA or Kruskal-Wallis test, depending on the assay. Differences were considered significant at p less than 0.05.
Statistical analysis was performed using the software GraphPad Prism version 6.0 for Windows, (GraphPad Software, San Diego, CA, USA).

Construction of plasmid for periplasmic purification
To overcome the problems associated with    Transformants were characterized by enzymatic digestion. The recombinant plasmid, pET26b/pilA, was extracted and its orientation confirmed by digestion with two restriction enzymes was mentioned above. The two expected bands were observed on gel: 649 and 5360 bp bands (Fig. 2).
Sequence analysis of recombinant pET26b/pilA confirmed that there were no amplification errors and that construction was accurate.

Expression and confirmation of the r-PilA
To construct an over-expression system, the coding sequence of the pilA gene, whose theoretical   To do this test, desired strains were incubated with different dilutions of mouse anti r-PilA antiserum and mouse macrophage in the presence of rabbit complement. The opsonic killing activity was observed when specific r-PilA antibody was treated with PAO1 and 6266E strains. No cross reaction was detected between NMS and the strains. Bars represent means of triplicate determinations, and error bar indicate SD.
Results were accepted to be significant at P less than 0.05. The Asterisks represents the groups which were significantly different (P < 0.05) and ND represents non-detectable difference.

Opsonophagocytic killing activity
We next performed an opsonophagocytic assay to test whether the r-PilA-specific antibodies present in the immunized mice sera could mediate killing of P. aeruginosa PAO1 and 6266E strains by macrophages in the presence of complement.
Unlike the NMS, the sera from mice immunized with the r-PilA showed opsonophagocytic killing activity against PAO1 and 6266E strains, to some extent, so that the number of viable bacterial cells decreased over 17.5% and 16.3% respectively, after 90 min compared with the control group (Fig. 5). In the presence of NMS, as the control group, no opsonic activity was observed. These data indicate that the anti r-PilA antibodies have a partial opsonic activity for killing of the P. aeruginosa strains.

Twitching inhibition assay
Immunized and non-immunized mouse sera were tested in the twitching inhibition assay to evaluate their functional activity for immobilization of P. aeruginosa strains PAO1 and 6266E. In this assay, NMS was used as control. As shown in Figure 6, the r-PilA antibody was able to inhibit slightly the motility of the PAO1 strain at a dilution colonization and potential invasion (17). A large number of published data showed that pili might be the most definitive antigen candidate (18,19). T4P are essential virulence factors of P. aeruginosa that have been extensively studied in the mouse model (8,20). Because of its length, the T4P has been associated with mediating of initial attachment of the bacteria to host surfaces before other adhesins secure the attachment. As soon as attachment occurs, the coordinated expression of other virulence factors facilitates invasion. Because of its early role in the pathogenesis of infection, the T4P has been suggested as an attractive vaccine target (21). A number of studies based on monoclonal antibody-binding data showed that the C-terminal DSL of the pilin subunit mediates attachment of bacteria to epithelial cell receptors (7,11 The most commonly used flexible linker has the sequence consisting of glycine and serine residues ("G/S" linker). An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser) n that has been used in this study.